Cholera toxin (CT), an enterotoxin expressed by Vibrio cholerae that is the virulence factor responsible for eliciting severe diarrheal symptoms in humans is released from the bacterium by a secretory mechanism encoded, in part, by chromosomal genes in the eps (extracellular protein secretion) cluster. Only CT and a few other periplasmic proteins (protease and chitinase) are secreted by the eps system, suggesting that specific transport signals recognized by the secretory apparatus are located in each protein. The PI and others have shown that V. cholerae secretes CT and LT-IIa, an enterotoxin produced by strains of Escherichia coli, with equal efficiency. Since the B polypeptides of CT and LT-IIa that are essential for extracellular transport share little, if any, amino acid homology, the transport signals on these toxins must be conserved structural motifs. Studies on the molecular aspects of toxin secretion by V. cholerae are hampered by the lack of a cloned secretion complex. Eps C, D, E, F, G, H, I, J, K, L, M, and N have been cloned, but the gene cluster does not confer upon E. coli the ability to secrete CT. These data suggest that additional genes are required to encode the secretion machinery. It is also likely that one or more of the Eps proteins interacts with CT on a physical level during transport. The PI's goals are: 1) to investigate the molecular structure of the extracellular transport signals in CT and LT-IIa; 2) to characterize the genetic lesion in CC9453, a mutant of V. cholerae that is deficient in extracellular secretion; and 3) to determine which of the Eps proteins have specific CT-binding activity. There is no longterm, effective vaccine for V. cholera. Until a vaccine is available, alternative strategies to ameliorate the symptoms of the disease are potentially very important. A better understanding of the process of extracellular secretion by V. cholerae is a necessary first step in the subsequent design of therapeutic strategies to ameliorate cholera symptoms by inhibiting release of CT.